The unique ability of hematopoietic stem cells to balance self-renewal with differentiation is crucial for development and homeostasis as well as for regeneration and repair. One fundamental way in which HSCs may be able to balance both self-renewal and differentiation is by utilizing a combination of asymmetric or symmetric division, differing modes of division mediated by the differential inheritance of cell fate determinants. In previous studies we have used live cell imaging to show that hematopoietic stem/progenitor cells can undergo both symmetric and asymmetric divisions. But how these types of divisions are controlled, and whether the loss of these mechanisms can affect cell fate and maintenance of the stem cell state are specific questions that remain to be answered. To address these issues, we have focused on Lis1, a dynein-binding protein that anchors the spindle to the cellular cortex and thereby regulates spindle orientation and correct inheritance of fate determinants in the nervous system. Using conditional knockout mice, we have found that loss of Lis1 leads to severe defects in the establishment of the hematopoietic system and a failure of self-renewal. Using real time imaging approaches that we have developed, we now propose to define whether the defects in hematopoietic stem cell fate in Lis1-deficient mice are linked to defects in asymmetric division as well as elucidate the downstream mechanisms important for Lis1 function. A better understanding of the mechanisms that regulate the balance between self-renewal and differentiation may enable development of new strategies to control hematopoietic stem cells and accelerate hematopoietic regeneration in times of need.